The present invention is related to the detection of protein in urine by the use of a test strip containing a protein error indicator and a buffer. More particularly, it relates to the use of a particular buffer, referred to herein as a "cation sensing buffer" which, when used in combination with typical buffer systems, helps to alleviate the result distorting effects which are observed when high specific gravity (SG) urine is tested for protein content in this manner.
The determination of the presence of protein in a urine sample is important in the diagnosis of several pathological conditions affecting the kidney and circulatory systems as well as the central nervous system. It is often necessary to qualitatively and quantitatively measure protein in urine. This is especially true in the diagnosis of diabetes and kidney disease. The predominant urine protein associated with diabetes is albumin which is the protein most commonly sought out in analyses.
Various methods for determining the presence of protein in urine are known, the most convenient of which involves wetting an absorbant test strip impregnated with a protein error indicator and buffer with a small quantity of urine. Protein error indicators are pH indicators which contain an ionizable group which is displaced in the presence of protein to provide a detectable color change. This is the same color change that the indicator would undergo under the influence of a pH change, so it is important to include buffer in the test strip to thereby avoid a pH increase since such an increase could cause the change of color in the indicator in the absence of protein thereby resulting in a false positive result.
The tendency toward false positive results is particularly problematical with the testing of high SG urine due to the presence in such urine of buffers which overwhelm the buffer in the test strip thereby permitting the change in pH resulting in the color change of the indicator which is indicative of the presence of protein when there is no protein present in the urine sample being tested. The primary buffering component in urine is phosphate, followed by citrate, uric acid, acetate, glycine and ammonium. This sort of false positive is, of course, to be avoided if at all possible. An example of such a false positive situation is a test for urine albumin in which the protein error indicator is Tetrabromophenol Blue, TBPB, which turns from yellow to blue above a pH of 3.7. Another common indicator, DIDNTB, turns from colorless to blue at a pH greater than 2.1. With the use of prior art buffers the addition of high SG urine, which typically has a pH of 5 to 8 and contains buffers which favor the maintenance of high pH thereby increasing the pH of the reagent system to a level of greater than 3.7, causes the color change which results in a false positive reading for protein in the urine sample.
In Japanese Patent Application No. 3-355044 there is described the use of potassium salts to reduce the tendency of false positive responses for protein in high SG urine. While it is stated on page 5 of this application that the mechanism is unclear, this system may operate on the principle that the potassium ion combines with a cation sensing component in urine even though the main components in urine are unresponsive cation sensing buffers. One of the potassium salts mentioned is potassium citrate. This reference also mentions the use of sodium citrate and citric acid as buffers. In example 15 of U.S. Pat. No. 5,279,790 there is described the preparation of a urine protein strip which involves impregnating a paper strip with a mixture of protein error indicators together with a potassium citrate buffer. Citric acid, like most anionic buffers possesses the ability to release some protons in the presence of metallic cations thereby lowering the pH in the presence of such cations. However in order for a cation sensing buffer to benefit the urine protein methodology, the buffer must release enough protons to lower the reagent pH when exposed to cations in the normal physiological ranges found in urine which, even in high SG urine, amount to a maximum of 140 mM for potassium and 250 mM for sodium. Other cations normally found in urine which contribute to this phenomena are calcium, magnesium and ammonium. Citric acid does not possess sufficient proton releasing ability to significantly lower pH at these cation concentrations. The highest urine buffering capacity is 35 meq/pH unit. A protein error indicator test should be resistant to this level of buffering capacity. A high pH (8-9) and a high buffering capacity urine can shift the pH of a citrate buffered reagent by as much as 0.5 units. The combined Na and K cation sensing ability should be greater than 0.5 pH units. The increase of 0.5 in reagent pH due to the buffering capacity of a high SG urine is offset by a decrease of 0.5 in reagent pH due to cation sensing ability of the buffer. The net result is little or no increase in reagent pH due to high SG urine, The combined Na and K cation sensing was 0.71 for tartaric acid and only 0.25 for citrate.